Role of transcription in genomic stability RNA polymerase (RNAP) sensitively detects damage as it performs one-dimensional scanning of template DNA, thus initiating the dedicated pathway of transcription-coupled repair (TCR). However, transcription can also increase mutagenesis. Hot spots for genomic instability implicated in human genetic disease and carcinogenesis have been localized in DNA sequences that can adopt non- canonical DNA structures (e.g. Z-DNA, H-DNA, G4-DNA, triplet repeats and palindromes forming hairpins/cruciforms). We wish to gain a mechanistic perspective on possible outcomes when RNAP encounters such structures. We hypothesize that TCR may be mutagenic when it occurs at or near non-canonical DNA structures, certain lesion types, or bound complex ligands. To test this hypothesis we will determine the precise signals that can arrest RNAP and elicit TCR, to learn whether TCR might be error-prone under some circumstances. An in vitro transcription assay using purified T7RNAP and mammalian RNAPII with required factors on defined DNA substrates will be utilized to: (1) Characterize RNAP arrested at site-specific non-canonical DNA structures and lesions, including psoralen monoadducts vs. interstrand crosslinks, and adducts of the acylfulvenes, which are reportedly subject to TCR but not global excision repair. The arrested RNAP, transcription bubble, and RNA/DNA hybrid will be mapped. Effects of added factors such as TFIIS, CSB and TFIIH, which may modulate transcription arrest, as well as effects of mismatch repair proteins and RecQ, which modulate some non B-form DNA structures will be determined. Recognition of non-canonical structures by repair enzymes in human cell extracts will be assessed (2) Evaluate cooperative effects on transcription of abasic sites or 8oxoGuanine introduced into Z-DNA, and effects of complex ligands, such as the Z-DNA binding protein ADAR1, and topoisomerase 1 trapped at abasic sites. (3) Utilize stable complexes of peptide nucleic acid (PNA) to explore novel aspects of these unique ligands for targeted gene alterations, while revealing mechanistic details of transcriptional processing. PNA binding will also be explored as an alternative to promoter-driven transcription, to possibly achieve higher efficiency of substrate usage, toward improved assays for transcription behavior at lesions and cell-free TCR assays. PUBLIC HEALTH RELEVANCE: The results from this project will enhance our understanding of the roles of transcription and TCR in processing lesions and other abnormalities in DNA that have been implicated in human disease. Since prolonged transcription arrest generates a strong signal for apoptosis, the research may lead to novel modes of chemotherapy, involving selective inhibition of TCR in target cells combined with administration of transcription-blocking drugs.